L-arabinose is a major constituent of plant material. L-arabinose fermentation is therefore also of potential biotechnological interest.
Fungi that can use L-arabinose and D-xylose are not necessarily good for industrial use. Many pentose utilising yeast species for example have a low ethanol tolerance, which makes them unsuitable for ethanol production. One approach would be to improve the industrial properties of these organisms. Another is to give a suitable organism the ability to use L-arabinose and D-xylose. There are pathways for D-xylose and L-arabinose, which are known to be active in bacteria. For D-xylose catabolism it is a xylose isomerase, which converts D-xylose to D-xylulose and a xylulokinase to make D-xylulose 5-phosphate. For L-arabinose catabolism the pathway consists of an isomerase, a kinase and an epimerase which convert L-arabinitol to L-ribulose, L-ribulose 5-phosphate and D-xylulose 5-phosphate, with D-xylulose 5-phosphate being an intermediate of the pentose phosphate pathway (Stryer, 1988). It has been tried to overexpress this bacterial pathway in the yeast S. cerevisiae, but it was not functional. The three enzymes of the L-arabinose pathway were expressed and shown to be active. However no growth on L-arabinose as a sole carbon source was reported (Sedlak and Ho, 2001). Also the expression of xylose isomerase in a fungal host was not successful (Sarthy et al. 1987, Chan et al. 1989, Kristo et al. 1989, Moes et al 1996, Schründer et al. 1996). The reason for this is not clear. There might be a species barrier, which prevents these bacterial isomerases from working in fungi. It can also be metabolic imbalances in the host, which are solved by an unknown mechanism in the donor.
There is also a hypothetical eukaryotic, i.e. fungal pathway, where L-arabinose is also converted to D-xylulose 5-phosphate, but by a different pathway (see FIG. 1). This pathway has been suggested to use 2 reductases, 2 dehydrogenases and a kikinase as shown (Chiang and Knight, 1961, Witteveen et al., 1989). While the genes of the bacterial pathway have been known for decades, very little is known about this hypothetical fungal pathway.
A fungal pathway for L-arabinose utilisation was described by Chiang and Knight (1961) for Penicillium chrysogenum and by Witteveen et al. (1989) for Aspergillus niger. It consists of an NADPH-linked reductase, which forms L-arabinitol, an NAD-linked dehydrogenase which forms L-xylulose, an NADPH-linked reductase which forms xylitol, an NAD-linked dehydrogenase which forms D-xylulose and a xylulokinase. The final product is D-xylulose 5-phosphate as in the bacterial L-arabinose pathway (see FIG. 1). This pathway was described only for filamentous fungi, but there are indications that it may also occur in yeast. Shi et al. (2000) described a mutant of Pichia stipitis which was unable to grow on L-arabinose. Over-expression of the NAD-linked xylitol dehydrogenase could restore the growth on L-arabinitol indicating that xylitol may be an intermediate in the L-arabinose pathway. Also yeast strains, which had L-arabinose as a sole carbon source, produced L-arabinitol and small amounts of xylitol (Dien et al., 1996), indicating that yeast might use this pathway. The capability of L-arabinose fermentation is not a common feature of yeast. Many yeast species mainly accumulate the L-arabinitol formed from L-arabinose (McMillan and Boynton 1994). Only recently yeast species were identified which were capable of L-arabinose fermentation (Dien et al., 1996).
The hypothetical fungal L-arabinose pathway has similarities to the fungal D-xylose pathway. In both pathways the pentose sugar goes through reduction and oxidation reactions where the reductions are NADPH-linked and the oxidations NAD-linked. D-xylose goes through one pair of reduction and oxidation reaction and L-arabinose goes through two pairs. The process is redox neutral but different redox cofactors, i.e. NADPH and NAD are used, which have to be separately regenerated in other metabolic pathways. In the D-xylose pathway an NADPH-linked reductase converts D-xylose into xylitol, which is then converted to D-xylulose by an NAD-linked de-hydrogenase and to D-xylulose 5-phosphate by xylulokinase. The enzymes of the D-xylose pathway can all be used in the L-arabinose pathway. The first enzyme in both pathways is an aldose reductase (EC 1.1.1.21). The corresponding enzymes in Saccharomyces cerevisiae (Kuhn et al. 1995) and Pichia stipitis (Verduyn, 1985) have been characterised. They are unspecific and can use either L-arabinose or D-xylose with approximately the same rate to produce L-arabinitol or xylitol respectively. Genes coding for this enzyme are known e.g. for Pichia stipitis (Amore et al., 1991), Saccharomyces cerevisiae (Kuhn et al., 1995, Richard et al. 1999), Candida tenius (Hacker et al., 1999), Kluyveromyces lactis (Billard et al., 1995) and Pachysolen tannophilus (Bolen et al., 1996).
The xylitol dehydrogenase (also known as D-xylulose reductase EC 1.1.1.9) and xylulokinase EC 2.7.1.17 are the same in the D-xylose and L-arabinose pathway of fungi. Genes for the D-xylulose reductase are known from Pichia stipitis (Kötter et al. 1990) Saccharomyces cerevisiae (Richard et al. 1999) and Tricoderma reesei (Wang et al. 1998). The gene for a fungal xylulokinase is only known for Saccharomyces cerevisiae (Ho and Chang, 1998)
Genes coding for L-arabinitol 4-dehydrogenase (EC1.1.1.12) or L-xylulose reductase (EC 1.1.1.10) are not known.
The invention aims to be able to express the pathway for L-arabinose utilisation in fungi. The hypothetical fungal pathway expressed in Saccharomyces cerevisiae would result in a strain, which can ferment nearly all sugars from forestry and agricultural waste to ethanol.